Gene delivery stent using titanium oxide thin film coating, and method for fabricating same

ABSTRACT

The present invention relates to a gene delivery stent using titanium oxide thin film coating and a method for fabricating the gene delivery stent. The gene delivery stent according to the present invention may be loaded with a drug having anti-inflammatory and anti-thrombotic effects and simultaneously deliver a gene capable of inhibiting proliferation of vascular smooth muscle cells. Accordingly, late thrombosis and metal allergy may be reduced, and vascular restenosis in the stent region may be prevented, thereby making it possible to increase treatment effects of the bare metal stent.

TECHNICAL FIELD

The present invention relates to a gene delivery stent using titaniumoxide thin film coating and a method for fabricating the same.

BACKGROUND ART

A bare metal stent is an implantable material used for treatment purposeso as to increase a blood vessel diameter by being grafted at a diseasesite at which the blood vessel diameter is reduced caused byatherosclerotic plaque, neoplastic tissue, cruor, or the like, whichblocks blood from flowing or allow the blood not to smoothly flow.

A disease in which this stent is mainly used is cardiovascular diseases,which account for 30% or more of causes of death in the world, and morethan 17.5 million people have died every year due to these diseases.Ischemic cardiovascular diseases occupying the largest portion of thecardiovascular diseases as described above mean that problems(atherosclerotic plaque, neoplastic tissue, or cruor) are generated in acoronary artery supplying blood to heart muscle, and a method fortreating ischemic heart disease is divided into a non-invasive method(drug therapy) and an invasive method (angioplasty). Among them, a baremetal stent used in conventional angioplasty, which is the invasivemethod, has an excellent treatment effect of enlarging a coronary arterydiameter to fix the enlarged coronary artery diameter, but it wasreported in academia and a clinical treatment field that restenosisoccurring after the bare metal stent was grafted was the biggestproblem.

A drug eluting stent (DES), which was developed in order to preventrestenosis as described above, significantly reduced a generation rateof restenosis in patents. However, in spite of incorporating thetreatment using an anti-thrombotic agent (aspirin and clopidogrel), latethrombus formation that did not appear well in animal tests has appearedin patents after 3 or 5 years or more of the graft of the drug elutingstent. As the reason, a hypothesis that the drug eluting stenteffectively inhibits proliferation or inflammation reactions of vascularsmooth muscle cells but excessively inhibits growth of endothelial cellsat the same time, and thus a re-endothelization process of covering asurface of the stent is delayed, which causes the late thrombusformation has been persuasively accepted until now.

For this reason, an effort to find an ideal method capable of notinhibiting growth of endothelial cells while inhibiting proliferation ofvascular smooth muscle cells has been continuously made domestically andglobally, but it is impossible to allow a drug used for the drug elutingstent to affect only a specific cell due to characteristics of achemical material. Therefore, as an alternative, a treatment methodusing genes that may be relatively cell-specifically expressed has beensuggested. In relation to this, various studies have been conducteddomestically and globally, and as a result, a method of preparing a DNAcontrolled release stent by coating a surface of the stent usingpolylactic-polyglycolic acid (PLGA) and collagen-polylactic-polyglycolicacid together with each other among non-degradable polymers to dischargea GFP gene, which is a report gene, to the outside of the stent has beenreported in 2000 (Gene Delivery from a DNA Controlled-Release Stent inPorcine Coronary Arteries, Bruce D. et al., NATURE BIOTECHNOLOGY, 2000,Vol. 18, 1181-1184; Bisphosphonate-Mediated Gene Vector Delivery fromthe Metal Surfaces of Stents, Ilia Fishbein et al., PNAS., 2006, Vol.103, 159-164).

In addition, an effect of delivering iNOS genes to reduce formation ofneointima by a method of coating a surface of a metal stent withpolyalanine bisphosphonate (PAA-BP), which is an aqueous polymer, andthen binding adenovirus thereto again using a binding agent was reportedby Ilia Fishbein et al., in 2006, such that a possibility of inhibitingrestenosis using the gene delivery stent was reported (DNA Delivery froman Intravascular Stent with a DenaturatedCollagen-Polylactic-Polyglycolic Acid-Controlled Release Coating:Mechanisms of Enhanced Transfection, I Perlstein et al., Gene Therapy,2003, Vol. 10, 1420-1428).

However, in most of the current technologies, in order to form a coatinglayer to which a gene complex may be bound on a surface of a metal sothat the gene complex is adhered to a metal stent made of stainlesssteel, a non-degradable organic polymer or degradable collagen has beenused, or in order to increase gene transfection efficiency, a viralvector has been used.

In the case of a technology of fabricating a gene eluting stent usingthe existing polymer, problems such as allergy, inflammation reactions,or the like, may be generated by the organic polymer, and it may besignificantly difficult to find a polymer having excellentbiocompatibility. In addition, in the case in which the stent isfabricated using the viral vector, it is still not free from apossibility of generating cancer and inducing immune reactions.

Therefore, introduction of another coating material for coating the genecomplex has been demanded. Particularly, this coating layer should havean appropriate functional group to which the gene complex may besufficiently bound, a uniform surface, and excellent biocompatibilitysuch as an anti-thrombotic property, an anti-inflammatory property, andthe like. In addition, only when a coated polymer layer has an excellentadhesive property with a substrate and excellent mechanical strength,the polymer thin film may endure a strong blood flow during a medicaloperation and disinfection process or after the medical operation for along period of time.

Therefore, in order to increase a success rate of introducing the DNAControlled-Release Stent, selection of a thin film material that doesnot have these problems and development of a coating technology thereofand a gene complex adhering technology are essentially demanded.

DISCLOSURE Technical Problem

Therefore, the present inventors confirmed an effect of reducing latethrombosis or metal allergy by coating a surface of an implantable metalstent with titanium oxide, modifying a surface of a titanium oxide thinfilm coated with titanium oxide to adhere drug and genes onto thesurface and an effect of preventing vascular restenosis in the stentregion by making it possible to transfect genes capable of inhibitinggrowth of cells into the cells, thereby completing the presentinvention.

An object of the present invention is to provide a gene delivery stentusing titanium oxide thin film coating capable of reducing latethrombosis or metal allergy and preventing vascular restenosis in thestent region by making it possible to transfect the gene capable ofinhibiting growth of cells into the cells, and a method for fabricatingthe same.

Technical Solution

In one general aspect, there is provided a method for fabricating a genedelivery stent using titanium oxide thin film coating capable ofreducing late thrombosis or metal allergy and preventing vascularrestenosis in a stent region by making it possible to transfect the genecapable of inhibiting growth of cells into the cells.

In another general aspect, there is provided a gene delivery stent usingtitanium oxide thin film coating fabricated by the method as describedabove.

The gene delivery stent according to the present invention may include:a titanium oxide thin film made of titanium dioxide (TiO₂),TiO_(2−x)N_(x) (0.001≦x≦1), or a mixture thereof and coated on a surfaceof a metal stent; a drug layer containing a drug having a function groupbound to a hydroxyl group of the titanium oxide thin film in which thehydroxyl group is introduced by modifying a surface of the titaniumoxide thin film to thereby be adhered onto the titanium oxide thin film;and an oligonucleotide layer containing oligonucleotide bound to thedrug to thereby be adhered onto the drug layer.

In more detail, the gene delivery stent may be fabricated by uniformlycoating the surface of the metal stent with the titanium oxide thin filmmade of titanium dioxide (TiO₂), TiO_(2−x)N_(x) (0.001≦x≦1), or themixture thereof at a thickness of 50 to 100 nm by a plasma enhancedchemical vapor deposition (PECVD) method, modifying the surface of thetitanium oxide thin film by a plasma process using water in order toadhere a sufficient amount of gene complexes to introduce a hydrophilicfunctional group into the coated surface of the titanium oxide thinfilm, separately adhere at least one drug selected from abciximab(ReoPro), heparin, and alpha-lipoic acid (ALA) thereto, and physicallyadhere the gene onto the surface of the drug.

The gene delivery stent according to the present invention may have atitanium oxide thin film/drug/gene complex structure by being fabricatedthrough the method as described above, wherein the complex structure hasadvantages in that a drug having anti-inflammatory and anti-thromboticeffects may be loaded and genes capable of inhibiting growth of vascularsmooth muscle cells may be simultaneously delivered.

Hereinafter, the present invention will be described in detail.

The present invention provides a method for fabricating a gene deliverystent using titanium oxide thin film coating, the method including:

-   -   1) a titanium oxide coating step of coating the surface of the        metal stent with titanium oxide represented by the following        Chemical Formula 1;    -   2) a surface modifying step of modifying the surface of the        coated titanium oxide thin film to introduce a hydroxyl group;    -   3) a drug adhering step of adhering the drug onto the titanium        oxide thin film surface-modified by binding the functional group        of the drug to the hydroxyl group of the titanium oxide thin        film to form the drug layer; and    -   4) an oligonucleotide adhering step of adhering oligonucleotide        onto the drug layer by binding oligonucleotide to the drug to        form the oligonucleotide layer. See FIG. 1.        TiO_(2−x)N_(x) (0≦x≦1)  Chemical Formula 1

The titanium oxide in the present invention may be titanium dioxide(TiO₂), TiO_(2−x)N_(x) (0.001≦x≦1), or the mixture thereof.

In the present invention, as the metal stent, a metal stent known in theart may be used without considering a shape, a length, a weight, or thelike. Preferably, a skeleton of the metal stent may have an elasticshape so that the shape is not changed by pressure in vessel and otherenvironmental affects while the metal stent stays in the vessel for along period of time and the stent has excellent mobility, and a materialthereof may be an anti-corrosive and harmless material. For example, astent disclosed in Korean Patent Laid-Open Publication No.10-2000-0069536, 10-1999-0035927, 10-1999-0087472, 10-2002-0093610,10-2004-0055785, or the like, may be used.

In detail, the metal stent may be preferably made of a biocompatiblemetal material such as stainless steel, nitinol, tantalum, platinum,titanium, cobalt, chromium, a cobalt-chromium alloy, acobalt-chromium-molybdenum alloy, and the like. In addition, otherbiocompatible metal material known in the art or a material bound to abiocompatible metal material may be used.

In the present invention, in the titanium oxide coating step (Step 1),the titanium oxide thin film may be coated onto the surface of the metalstent for 1 to 6 hours by transferring a titanium precursor, carriergas, and reaction gas and generating plasma in a plasma vacuum chamber.

The titanium precursor may be at least one selected from a groupconsisting of titanium butoxide, tetra-ethyl-methyl-amino-titanium,titanium ethoxide, titanium isopropoxide, andtetra-methyl-hepta-diene-titanium, and any titanium precursor known inthe art may be used as long as the precursor has excellent depositioncharacteristics at the time of depositing the titanium oxide onto themetal stent using the plasma enhanced chemical vapor deposition method.

In the present invention, as the carrier gas, at least one selected froma group consisting of nitrogen, argon, and helium may be used, and asthe reaction gas, at least one selected from water vapor, ozone, andoxygen may be used. Here, nitrogen-doped titanium oxide may be formed byadding nitrogen to the reaction gas.

More specifically, in the present invention, the titanium oxide may bedeposited on the surface of the metal stent using the titanium precursorby the plasma enhanced chemical vapor deposition method. In this case, aplasma enhanced chemical vapor deposition apparatus known in the art maybe used. For example, a plasma enhanced chemical vapor depositionapparatus disclosed in Korean Patent Application Nos. 10-2005-0058926,10-2001-0007030, 10-1990-0013643, or the like, may be used. Preferably,as shown in FIG. 2, a plasma enhanced chemical vapor depositionapparatus having a shape in which a titanium wire G capable of fixing astent H in a plasma vacuum chamber is installed, a reduced pressure pumpB is connected to the plasma vacuum chamber, a bubbler C filled with aprecursor and gas tanks E and F are connected so that titanium precursorvapor may be injected into the plasma vacuum chamber together with gas,and the bubbler is connected to another gas tank D may be used.

Before the titanium oxide is deposited onto the metal stent, a processof fixing the metal stent into the plasma vacuum chamber andpre-treating the surface of the metal stent to wash the surface may beperformed. The pre-treating process is performed in order to improvedeposition strength between the stent and the titanium oxide thin filmby maintaining a temperature in the plasma vacuum chamber at 200 to 600°C. and flowing argon and oxygen mixed gas.

In the present invention the titanium oxide of [Chemical Formula 1]deposited onto the metal stent may have two forms. One may be titaniumdioxide (TiO₂) formed by binding between titanium and oxygen, and theother may be TiO_(2−x)N_(x) (0.001≦x≦1) in which titanium dioxide (TiO₂)formed by binding between titanium and oxygen is doped with nitrogen.

A crystalline structure of the titanium oxide is not considered. Forexample, the crystalline structure may be a rutile form, an anataseform, a brookite form, or the like. The titanium oxide thin film iscoated onto the surface of the metal stent by introducing the carriergas in the chamber and the titanium precursor in order to deposit thetitanium oxide thin film coating onto the metal stent using the plasmaenhanced chemical vapor deposition method and generating plasma whileflowing the reaction gas. In this case, the two forms of the titaniumoxide may be determined according to a kind and flux ratio of theflowing reaction gas. Here, the carrier gas and the titanium precursormay be introduced into the plasma vacuum chamber by flowing at least onegas selected from the group consisting of nitrogen, argon, and helium asthe carrier gas together with the titanium precursor. At the same time,in the case of generating the plasma while flowing only oxygen, titaniumdioxide is deposited onto the surface of the metal stent, and in thecase of generating plasma while flowing oxygen together with nitrogen,nitrogen-doped titanium oxide (TiO_(2−x)N_(x) (0.001≦x≦1)) is depositedonto the surface of the metal stent.

Therefore, in order to deposit titanium dioxide (TiO₂) onto the metalstent, at the time of selecting the carrier gas, the carrier gas thatdoes not include nitrogen may be preferable. It was proven that titaniumoxide was biologically and biochemically stable as described above, andin the case of nitrogen-doped titanium oxide (TiO_(2−x)N_(x)(0.001≦x≦1)), the fact that when titanium dioxide is doped withnitrogen, the anti-thrombotic effect was further improved was alreadyreported by Kastarti A. and 12 others (Kastarti A., Mehilli J., PacheJ., Kaiser C., Valgimigli M., Kelbaek H., Menichelli M., Sabate M.,Suttorp M. J., Baumgart D., Seyfarth M., Pfisterer M. E., Schomig A., N.Engl. J. Med., 356, 1030, 2007).

According to the present invention, in order to introduce the titaniumprecursor into the plasma vacuum chamber, it may be preferable togenerate gaseous titanium precursor using the bubbler and then introducethe gaseous titanium oxide. Here, the gaseous titanium precursor may betransferred into the plasma vacuum chamber by pre-heating the bubbler ata temperature suitable for generating the vapor phase in a range of roomtemperature to a boiling point of the titanium precursor and allowingthe carrier gas to pass through the bubbler. At this time, titaniumdioxide and nitrogen-doped titanium oxide (TiO_(2−x)N_(x) (0.001≦x≦1))may be formed by mixing oxygen or oxygen and nitrogen together with thecarrier gas to transfer them. When the titanium precursor, the carriergas, and the reaction gas are introduced into the plasma vacuum chamber,the plasma may be generated in the plasma vacuum chamber to thereby bechemically deposited onto the surface of the metal stent. In this case,the flux of the carrier gas may be 50 to 500 sccm, preferably 100 to 200sccm, and the reaction gas may be injected at a flux of 10% of that ofthe carrier gas, preferably, 10 to 100 sccm. Further, in the case inwhich the reaction gas is not only oxygen but a mixed gas formed ofdifferent gases, that is, oxygen and nitrogen, oxygen and nitrogen maybe injected at a flux ratio of 1 to 9:9 to 1.

According to the present invention, the titanium oxide may be depositedonto the surface of the metal stent by introducing the titaniumprecursor, the carrier gas, and the reaction gas and generating theplasma in the plasma vacuum chamber. A discharge power of the plasma maybe 1 to 300 W and a reaction time may be 1 to 6 hours. Preferably, theplasma discharge may be performed at 5 to 200 W for 3 to 5 hours.

In the titanium oxide thin film coated-metal stent obtained after theabove-mentioned process, a thickness of the titanium oxide thin film maybe 10 to 500 nm, preferably 50 to 100 nm.

According to the present invention, in the surface modifying step (Step2), the surface of the titanium oxide thin film may be modified for 10minutes to 2 hours by transferring water vapor, or oxygen and hydrogeninto the plasma vacuum chamber and generating non-thermal plasma.

According to the present invention, as the drug in step 3), at least oneof abciximab, ALA and heparin may be selected and adhered.

More specifically, in the metal stent coated with the titanium oxidethin film by the plasma enhanced chemical vapor deposition method, inorder to adhere the drug to the surface of the titanium oxide thin film,the surface of the titanium oxide thin film may be modified in a form inwhich the hydroxyl group (—OH) is introduced. The surface modificationmethod may be performed in the plasma vacuum chamber used for formingthe titanium oxide, and water vapor (H₂O) is transferred from anexternal introduction tube connected to the plasma vacuum chamber intothe plasma vacuum chamber at a reduced pressure of 1×10⁻³ to 1 torr,preferably 1×10⁻² to 1×10⁻¹ torr. Alternatively, mixed gas of hydrogenand oxygen may be used instead of water vapor. In this case, a flux ofthe water vapor or the mixed gas of hydrogen and oxygen may be 1 to 50sccm based on a stent unit body. When the water vapor or the mixed gasof hydrogen and oxygen is introduced into the plasma vacuum chamber andthe plasma is generated, oxygen in the surface of the titanium dioxidelayer or the nitrogen-doped titanium oxide (TiO_(2−x)N_(x) (0.001≦x≦1))may be modified into the hydroxyl group.

At this time, the surface of the titanium dioxide layer or thenitrogen-doped titanium oxide (TiO_(2−x)N_(x) (0.001≦x≦1)) layer may bemodified into the surface in which the hydroxyl group is introduced bycarrying out the reaction at a discharge power of the plasma of 1 to 300W for 10 minutes to 2 hours, as a reaction condition.

The reason for modifying the titanium oxide thin film coated onto themetal stent into the surface in which the hydroxyl group is introducedas follow. In views of chemical structural characteristics, since thedrugs of the present invention have a functional group such as acarboxyl group, an aldehyde group, an alcohol group, or the like, it maybe easy to bind the drug to the surface of the titanium oxide thin filmthrough a dehydrogenation reaction by chemically reacting with thehydroxyl group in the surface of the modified titanium oxide thin filmunder an acidic condition.

In addition, in the drug and the titanium oxide thin film that are boundto each other by the principle as described above, after the drug isbonded thereto, various functional groups in the drug may be physicallybonded to the surface of the stent to thereby be adhered to the surfaceof the stent in several layers. Therefore, at the time of inserting thedrug eluting stent into blood vessel in body, the drug may bedelayed-released while the physically bonded drug is separated from thedrug eluting stent, and since the titanium oxide, and the drug maymaintain their original structures, the drug may stably remain on thesurface of the metal after the release of the drug is completed.

In the present invention, the drug means a drug capable of inhibitingneointimal hyperplasia or thrombus adhesion, and an example thereof mayinclude at least one drug selected from a group consisting ofanti-cancer drugs, anti-inflammatory drugs, smooth muscle cell growthinhibitors, anti-thrombotic agents, and the like.

In more detail, at least one drug selected from molsidomine,linsidomine, nitroglycerin, hydralazine, verapamil, diltiazem,nifedipine, nimodipine, captopril, enalapril, lisinopril, quinapril,losartan, candesartan, irbesartan, valsartan, dexamethasone,betamethasone, prednisone, corticosteroid, 17-beta-estradiol,cyclosporine, mycophenolic acid, tranilast, meloxicam, Cerebrex,indomethacin, diclofenac, ibuprofen, naproxen, serpin, hirudin, hirulog,argatroban, sirolimus, rapamycin, rapamycin derivatives, paclitaxel,7-hexanoyl-taxol, cisplatin, vinblasitne, mitoxantrone, combretastatinA4, topotecan, methotrexate, flavopiridol, actinomycin, ReoPro(abciximab), alpha lipoic acid, heparin, warfarin, aspirin, abiprofen,prostaglandin, and the like, may be used. Preferably, at least oneselected from heparin, Reopro (abciximab), alpha lipoic acid, sirolimus,rapamycin, actinomycin, molsidomine, linsidomine, paclitaxel, and thelike, may be used.

That is, as the drug adhered to the titanium oxide thin film, alphalipoic acid (ALA) having the anti-inflammatory effect or abciximab andHeparin having the anti-thrombotic effect are used, such that theanti-inflammatory effect and the anti-thrombotic effect may be obtainedat the time of grafting the stent in body.

One of these drugs may be physically and chemically bonded to thesurface of the titanium oxide thin film to release the drug in the body,or at least two different kinds of drugs may be independently bonded tothe surface of the titanium oxide thin film to release at least twokinds of drug, thereby fabricating a multiple drug eluting stent.

If the drug is a mixture of at least two kinds of drugs, each of thedrugs may be directly dispersed onto the surface-modified titanium oxidethin film to be bonded to the surface, or at least two kinds of drugsare physically and chemically bound to each other by electrostaticinteraction between drugs to be bonded to the titanium oxide thin film,hydrogen bond, or the like, to thereby be released in the body See FIG.3. For example, when lipoic acid and ReoPro are bound to the titaniumoxide thin film together with each other, since lipoic acid has theanti-inflammatory effect but lacks the anti-thrombotic effect, theanti-thrombotic effect may be improved by ReoPro.

In the present invention, in the drug adhering step (step 3), theobtained metal stent including the titanium oxide thin film in which thehydroxyl group is introduced in the surface modification step (step 2)may be injected into an independent reactor known in the art and mixedwith the drug, deionized water or an organic solvent may be furtherinjected thereto, as needed, followed by stirring in an acidic solutionunder inert atmosphere, thereby performing the reaction.

In the present invention, the oligonucleotide in step 4) may includeDNA, RNA, and a synthetic isoform thereof. In more detail, theoligonucleotide may be selected from a group consisting of genomic DNA(gDNA), complementary DNA (cDNA), plasmid DNA (pDNA), messenger RNA(mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), small interfering RNA(siRNA), micro RNA (miRNA), and antisense-oligonucleotide.

The oligonucleotide may exist in nature or be synthesized and derivedfrom human, animals, plants, bacteria, virus, or the like. Theoligonucleotide may be obtained by a method in the art.

In the present invention, in the oligonucleotide adhering step (step 4),a functional group of the oligonucleotide may be adhered to thefunctional group of the drug by at least one bond of a hydrogen bond, adipole-dipole bond, an induced dipole bond, and a disulfide bond (S—Sbond) therebetween.

The oligonucleotide layer adhered onto the drug layer has advantages inthat the oligonucleotide may be efficiently delivered into cells by thebond and the proliferation of the vascular smooth muscle cells may beinhibited without adverse effects such as thrombus, inflammation, andthe like.

The present invention provides a gene delivery stent using titaniumoxide thin film coating, the gene delivery stent including: a titaniumoxide thin film obtained by coating a surface of a metal stent withTiO₂, TiO_(2−x)N_(x) (0.001≦x≦1), or a mixture thereof and modifying thecoated surface to introduce a hydroxyl group; a drug layer containing adrug having a functional group bound to a hydroxyl group of the titaniumoxide thin film to thereby be adhered onto the titanium oxide thin film;and an oligonucleotide layer containing oligonucleotide bound to thedrug to thereby be adhered onto the drug layer.

In the gene delivery stent according to the present invention, thetitanium oxide thin film may be the titanium dioxide (TiO₂) thin film orthe nitrogen-doped titanium oxide (TiO_(2−x)N_(x) (0.001≦x≦1)) thinfilm.

In the gene delivery stent according to the present invention, the drugmay be at least one of abciximab, ALA, and heparin.

In the gene delivery stent according to the present invention, theoligonucleotide may be selected from a group consisting of gDNA, cDNA,pDNA, mRNA, tRNA, rRNA, siRNA, miRNA, and antisense-oligonucleotide.

Advantageous Effects

The gene delivery stent using titanium oxide thin film coating accordingto the present invention is fabricated by coating the surface of theimplantable metal stent with the titanium oxide, modifying the surfaceof the coating layer, and adhering the drug and the gene to the modifiedsurface, such that the titanium oxide thin film and theanti-inflammatory or anti-thrombotic drug may stably remain in a surfaceof the metal even though elution of the gene and the drug is completed,thereby reducing late thrombosis and metal allergy. In addition, thegene capable of inhibiting growth of cells may be transfected into thecell, thereby preventing vascular restenosis in the stent region.

The gene delivery stent using titanium oxide thin film coating accordingto the present invention fabricated by the above-mentioned method may beloaded with a drug having anti-inflammatory and anti-thrombotic effectsand at the same time, deliver a gene capable of inhibiting proliferationof vascular smooth muscle cells. Accordingly, late thrombosis and metalallergy may be reduced, and vascular restenosis in the stent region maybe prevented, thereby making it possible to increase treatment effectsof the bare metal stent.

DESCRIPTION OF DRAWINGS

FIG. 1 is a view showing a process of sequentially coating a titaniumoxide thin film and a drug on a surface of a metal stent and coatingoligonucleotide thereon again in a gene delivery stent using titaniumoxide thin film coating according to an exemplary embodiment of thepresent invention;

FIG. 2 is a mimetic view of a PECVD apparatus for coating titaniumdioxide or nitrogen-doped titanium oxide (TiO_(2−x)N_(x) (0.001≦x≦1))used in the gene delivery stent using titanium oxide thin film coatingaccording to the exemplary embodiment of the present invention;

FIG. 3 is a mimetic view of a state in which alpha-lipoic acid (ALA) isadhered to the titanium oxide thin film in the gene delivery stent usingtitanium oxide thin film coating according to the exemplary embodimentof the present invention;

FIG. 4 is graphs obtained by electron spectroscopy for chemical analysis(ESCA) of a TiO₂ thin film deposited at 5 W and 400° C. for 4 hours inthe gene delivery stent using titanium oxide thin film coating accordingto the exemplary embodiment of the present invention.

FIG. 5 is graphs obtained by ESCA of a nitrogen-doped TiO₂ thin filmdeposited at 5 W and 400° C. for 4 hours in the gene delivery stentusing titanium oxide thin film coating according to the exemplaryembodiment of the present invention;

FIG. 6 is a graph showing a contact angle change according to adischarge power applied to surface modification in the gene deliverystent using titanium oxide thin film coating according to the exemplaryembodiment of the present invention;

FIG. 7 is a simple mimetic view of a plasmid allowing whether or not thegene is transfected to be known by generating beta-galactosidase as aplasmid coated in the gene delivery stent using titanium oxide thin filmcoating according to the exemplary embodiment of the present invention;

FIG. 8 is a graph showing an amount of genes coated on each of threedrug (abciximab, heparin, ALA) coating layers in the gene delivery stentusing titanium oxide thin film coating according to the exemplaryembodiment of the present invention;

FIG. 9 is photographs showing that the genes are safely coated on atitanium oxide/Abciximab/plasmid composite layer, a titaniumoxide/Heparin/plasmid composite layer, and a titanium oxide/ALA/plasmidcomposite layer without damage in functions of the gene, respectively,and all of the genes (g-Wiz lacZ plasmid) are normally expressed incells, in the gene delivery stent using titanium oxide thin film coatingaccording to the exemplary embodiment of the present invention;

FIG. 10 is photographs showing that the genes are transfected intotissues after metal pieces coated with each of the titaniumoxide/Abciximab/plasmid composite layer, the titaniumoxide/Heparin/plasmid composite layer, and the titaniumoxide/ALA/plasmid composite layer are grafted in bodies of rats for anexperiment, in the gene delivery stent using titanium oxide thin filmcoating according to the exemplary embodiment of the present invention;

FIG. 11 is cross-sectional views of the tissues into which the genes aretransfected after the metal piece coated with each of the titaniumoxide/Abciximab/plasmid composite layer, the titaniumoxide/Heparin/plasmid composite layer, and the titaniumoxide/ALA/plasmid composite layer are grafted in bodies of rats for theexperiment, in the gene delivery stent using titanium oxide thin filmcoating according to the exemplary embodiment of the present invention;and

FIG. 12 is a photograph showing that when porcine coronary vascularsmooth muscle cells are cultured on the metal pieces coated with thetitanium oxide/Abciximab/plasmid composite layer, the genes aretransfected into the cells.

BEST MODE

-   -   (1) Titanium oxide thin film coating on surface of metal

A metal plate having a size of 1 cm×1 cm was fabricated in a disk shapeusing stainless steel was fabricated among materials used for a stent,and titanium oxide thin film coating was performed on a surface of themetal plate.

The metal plate was fixed to a plasma generator as a stent H shown inFIG. 2 in a vacuum chamber connected to a radio frequency (RF) plasmagenerator generating plasma and a vacuum pump, and a temperature of theplasma vacuum chamber was maintained at 400°. Firstly, in order toimprove adhesion between a substrate of the metal plate and a thin film,the surface of the metal was subjected to a plasma pre-treatment processby flowing argon and oxygen before thin film coating to wash the surfaceof the metal. Titanium isopropoxide was put into a bubbler evaporator,mixed with oxygen, which was reaction gas using argon (Ar), which wascarrier gas while maintaining a temperature of the bubbler at 50°, andthen introduced in a reaction chamber, followed by generating plasma toperform a reaction for 4 hours, thereby coating the surface of the metalplate with a titanium dioxide thin film. In this case, a flow rate ofargon (Ar), which was the carrier gas, was 100 sccm, and a flow rate ofoxygen, which was the reaction gas, was maintained at 20 sccm. Dischargepower was variously applied in a range of 5 to 200 W to coat the thinfilm. In order to fabricate a nitrogen-doped titanium dioxide thin film,the above-mentioned experimental conditions were equally maintainedexcept that argon, oxygen, and nitrogen were supplied at flow rates of100 sccm, 10 sccm, and 1 sccm, respectively.

The discharge power may be variously applied from 5 to 200 W at the timeof coating the titanium dioxide thin film, but it was confirmed that asthe larger the discharge power, the higher the surface roughness. Rootmean square (Rms) values of results obtained by an atomic forcemicroscope (AFM) of the thin film fabricated for 4 hours while changingdischarge power at 5, 10, and 15 W were shown in the following Table 1.

TABLE 1 Sample NT5 NT10 NT25 T5 T10 T15 Rms 3.571 5.142 7.119 7.7609.403 13.862 T: titanium dioxide coated thin film, NT: nitrogen-dopedtitanium dioxide coated thin film, numbers (5, 10, 15): discharge powerapplied at the time of depositing titanium dioxide thin film

As shown in Table 1, it was confirmed that in the case of thenitrogen-doped thin film, the surface was more uniform than in the caseof the titanium dioxide thin film and the lower the discharge power, themore uniform the obtained thin film. It was known that the roughness ofthe thin film affects blood compatibility, and as the surface roughnessis reduced, blood compatibility becomes excellent.

In addition, the thin film fabricated at 5 W had the lowest surfaceroughness. Therefore, all of the titanium dioxide thin film depositionfor surface modification was performed while fixing discharge power at 5W and maintaining a temperature 400° C. for 4 hours, thereby fabricatinga stent coated with titanium dioxide.

In FIG. 4 showing results of ESCA of the titanium dioxide thin filmfabricated at 5 W, peaks corresponding to Ti⁴⁺ in TiO₂ were confirmed at458.8 eV (2p3/2) and 464.7 eV (2p1/2), and an O1s peak corresponding toa Ti—O bond in TiO₂ was confirmed at 530.4 eV.

In FIG. 5 showing results of ESCA of the nitrogen-doped titanium dioxidethin film, Ti peaks and O1s peaks were confirmed at positions similar tothat in the titanium dioxide thin film, and an N1s peak was confirmed at399 eV at a content ratio of 0.8%, such that it was confirmed that thesurface of the titanium dioxide was doped with nitrogen.

-   -   (2) Modification of titanium oxide thin film coating layer for        generating hydroxyl group

In order to adhere the drug onto the surface of the coated titaniumdioxide, a functional group capable of chemically binding to afunctional group in drug molecules needs to exist in the surface of thetitanium oxide.

Therefore, in the present invention, in order to introduce —OH group inthe surface of the titanium dioxide layer capable of chemically bindingto the drug, the surface of the thin film was modified by non-thermalplasma using deionized water (H₂O). After the metal plate coated withtitanium dioxide was fixed to a tubular non-thermal plasma reactor madeof Pyrex and the bubbler was filled with tertiary deionized water, watervapor was introduced into the plasma reactor at 10 sccm and dischargepower was changed in a range of 10 to 100 W, thereby modifying thetitanium dioxide thin film for 10 minutes using a non-thermal plasmaprocess.

In order to introduce the hydroxyl group in the surface of the titaniumdioxide thin film, after the surface was modified using H₂O, the surfacewas washed with deionized water once, and then a contact angle wasmeasured. Results between the discharge power and the contact angleapplied for modification were shown in FIG. 6.

As a result, as shown in FIG. 6, entirely, the contact angle was reducedby about 40 (degrees) than the contact angle before modification, it wasconfirmed that a hydrophilic functional group was introduced in thesurface. In addition, it was confirmed that the contact angle wasreduced when the applied discharge power was in a range of 20 to 50 W,and as the discharge power became higher than 60 W, there was anincreasing tendency in contact angle.

This may be because as the discharge power was increased, the titaniumdioxide thin film, which was a target for modification, was slightlyetched by the plasma, or a structure of titanium dioxide was changed.

-   -   (3) Drug adhesion

The used drugs were as follows.

-   -   (1) α-lipoic acid (Thiocticaid (ALA); Bukwang Pharm. Co. Ltd)    -   (2) Abciximab (ReoPro; Eli Lilly and Company, Indianapolis,        Ind.)    -   (3) Heparin sodium salt (Grade1-A, From Porcine Intestinal        Mucosa, Sigma-Aldrich)    -   (4) 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide methiodide        (DCC; 98%, Alfa Aesar)    -   (5) 4-(Dimethylamino)pyridine (DMAP; 99%, Sigma-Aldrich Co.)    -   (6) Sodium bicarbonate (99%, Dae Jung Chemicals & Metals Co.,        Ltd)

In order to adhere ALA onto the surface of the titanium dioxide coatinglayer, 0.0124 g of ALA, 0.005 g of sodium bicarbonate were sufficientlydissolved by adding 2 mL of deionized water, respectively, to prepare anALA solution, 0.8915 g of DCC and 0.07 g of DMAP were dissolved in 150mL of deionized water, respectively. 2 mL of the prepared ALA solutionand 1.5 mL of the DMAP+DCC mixed solution were harvested, respectively,and put into a vessel, followed by mixing. Then, the mixed solution wasleft at 35° C. for 1 hour to activate the mixed solution, and thesurface-modified metal plate fabricated in Surface-modified metal platewas put into the activated mixed solution and stored at 35° C. for 2hours, thereby adhering the drug to the surface of the titanium dioxidelayer.

Heparin was adhered by the same method except that 1.5 mL of theprepared DMAP+DCC mixed solution was put into 0.0065 g of heparin sodiumsalt.

Abciximab was adhered by the same method except that 1.5 mL of theDMAP+DCC mixed solution was put into 0.25 mL of abciximab.

-   -   (4) Gene adhesion

The drug adhered to the surface of the metal plated coated with titaniumdioxide had various kinds of chemical functional groups. For example,there were a carboxyl group, an amine group, a disulfide (S—S) bond, andthe like. A plasmid was put into an aqueous solution maintained at a pHof 6 to 7, and the stent containing the drug adhered thereto was addedthereto and maintained at room temperature for a predetermined time (1to 5 hours). In this case, various physical interactions were generatedbetween various functional groups in the drug adhered to the surface ofthe metal plate coated with the titanium dioxide thin film andfunctional groups existing in the plasmid, such that the plasmid wasadhered to the surface of the metal plate in several layers.

In order to confirm whether or not the gene was delivered from the metalplate composed of the titanium dioxide/drug/gene complex fabricated bythe above-mentioned method, gWIZ-β-gal plasmid was purchased fromGenlantis Company. When gWIZ-beta gal plasmid is delivered into thecells, the cells generate β-Galactosidase, wherein β-Galactosidase,which is an enzyme hydrolyzing β-galactoside, is used as a reporter genein eukaryotic transfection experiment. In cells in which the gene istransfected, β-Galactosidase cleaves5-bromo-4-chloro-3-indoyl-beta-D-galactopyranoside (X-gal) to generateblue precipitates. Since this blue color may be observed in tissue orcell through a microscope or by the naked eyes, it is possible todetermine whether the gene was transfected or not.

FIG. 7 shows a simple mimetic view of the plasmid allowing whether ornot the gene is transfected to be known by generating beta-galactosidaseas gWIZ-β-gal plasmid.

Experimental Example 1 Quantification of Adhered Gene

The metal plate fabricated by the methods in Examples (1) to (3) waspositioned on a 12-well plate, and a solution containing genes (totalplasmid content: 20 ug/200 ul DW) was put onto the metal plate. Afterthe metal plate was left for 8 to 12 hours, the metal plate was immersedagain in sterile deionized water (DW) for 30 minutes to remove extra DNAnon-specifically adhered thereto. A concentration of the plasmid inwashing DW was measured using Nanodrop ND-1000 spectrophotometer (Thermoscientific, USA). After the plate was washed and dried in a sterilebench, subsequent experiments were performed. The amount of DNA adheredto the flask was estimated by arithmetically subtracting a measuredamount of the DNA in the washing DW from initial 20 ug of plasmidaccording to the following Equation 1.DNA binding amounts (ug)=20 ug−DNA amount (ug) in washing DW  Equation 1

FIG. 8 is a graph showing the amount of the gene coated on each of threedrug (abciximab, heparin, ALA) coating layers in the gene delivery stentusing titanium oxide thin film coating according to the exemplaryembodiment of the present invention. In FIG. 8, it may be confirmed thatin the case of a TiO₂ single coating group, in the case of an abciximabcoating group, in the case of a heparin coating group, and in the caseof ALA coating group, amounts of plasmid coated at an area of 1 cm³ wereapproximately 2.7 ug, 7.2 ug, 5.6 ug, and 2.5 ug, respectively.

Experimental Example 2 Intracellular Expression of Gene Eluted fromMetal/Titanium Dioxide/Drug/Gene Complex

In order to confirm whether or not functional deformation was generatedin the plasmid after the gene was bound to the surface of the metalplate in a form of titanium/drug/gene (plasmid) complex (hereinafter,referred to as a metal flask), after the metal flask was generated, thegene was artificially separated, followed by measuring whether or notthe gene had an activity.

In order to elute the gene from the metal flask, the metal flask was putin a 12-well plate, and 100 uL of 0.1×TE buffer (pH 8.0) was addedthereto again and left at room temperature for 30 minutes. After 30minutes, 0.1×TE buffer was harvested, and an amount of the elutedplasmid was measured using Nanodrop ND-1000 spectrophotometer (ThermoScientific, USA).

In order to confirm whether or not the eluted plasmid was normallyactive, the gene was transfected in cells under in vivo conditions,using Lipofectamine 2000, which is a transfection product fabricated byInvitrogen Company. As described below, human embryonic kidney 293 Tcells (HEK 293 T cell) were seeded in a 12-well plate at 1×10⁵/well andcultured. After 24 hours, a culture medium was treated with 6 ug of theeluted plasmid using Lipofectamine, and after 4 hours, the culturemedium was stirred, followed by continuously culturing for 48 hours.Then, X-gal staining was performed.

After fixing the cell, the cell was stained with an X-gal stain solutionat 37° C. for 24 hours. Thereafter, in the case of observing whether thecell was stained or not, the positively stained cell was observed as ablue stained cell in an optical microscope.

As a result, as shown in FIG. 9, it may be confirmed that the genes weresafely coated on each of the gene delivery stents using titanium oxidethin film coating according to the exemplary embodiment of the presentinvention, that is, the titanium dioxide/abciximab/plasmid compositelayer, the titanium dioxide/heparin/plasmid composite layer, and thetitanium dioxide/ALA/plasmid composite layer without damaging thefunctions of the gene, and all of the genes (g-Wiz lacZ plasmid) werenormally expressed in cells.

In addition, as shown in FIG. 9, it was confirmed that all of theplasmid eluted from abciximab, ALA, and heparin normally allowed thecell to generate β-galactosidase. As the result, it may be confirmedthat the functions of the gene coated in the present invention weremaintained.

Experimental Example 3 Confirmation of Whether or Not Genes areDelivered in Abdominal Wall of Rats for Experiment

The metal flask fabricated in the Examples was grafted in abdominal wallof rats for the experimental. After 7 days of grafting, muscles of theabdominal wall were harvested and the X-gal staining was performed bythe same method as that in Experimental Example 2. After staining, themuscle in abdominal wall was cut, and a stained site was confirmed.

FIG. 10 is a photograph showing that the genes were transfected intotissues after the gene delivery stent using titanium oxide thin filmcoating according to the exemplary embodiment of the present invention,that is, metal pieces coated with each of the titaniumdioxide/abciximab/plasmid composite layer, the titaniumdioxide/Heparin/plasmid composite layer, and the titaniumdioxide/ALA/plasmid composite layer were grafted in bodies of the ratsfor the experiment.

As shown in FIG. 10, it may be confirmed that the sites at which thetissue in the abdominal wall was stained as a blue color were observedby the naked eyes. The results shows that the β-gal gene-coated platesuccessfully transfected the gene in the abdominal tissue.

In addition, as shown in FIG. 11, it may confirmed that the cells of thetissue in which the gene was transfected by grafting the metal piececoated with each of the titanium dioxide/abciximab/plasmid compositelayer, the titanium dioxide/Heparin/plasmid composite layer, and thetitanium dioxide/ALA/plasmid composite layer, in the body of the ratsfor the experiment delivered the gene to a connective tissue (red arrow)on the abdominal wall, and at the same time, the stained sites were alsoobserved in the tissue cells (yellow arrow) under the connective tissueby the microscope.

Experimental Example 4 Confirmation of Whether or Not Genes areDelivered in Vascular Smooth Muscle Cells

In order to confirm whether or not the gene may be transfected in thevascular smooth muscle cell capable of being a main graft site of thestent besides the connective tissue and the muscle tissue from the platecoated with the gene as confirmed in Experimental Example 3, thevascular smooth muscle cells were directly cultured in the surface ofthe metal plate, followed by confirming whether the gene was transfectedin the cell.

The vascular smooth muscle cells were separated from the porcinecoronary artery separated in a sterile state. After separating thecoronary artery from a heart of the pig, all of the connective tissuesin the outside of the blood vessel were removed, and vascularendothelial cells were removed by scratching the inside of the bloodvessel using forceps. The blood vessel tissue from which the connectivetissue and the endothelial cell were removed was put into a solutioncontaining collagenase, elastase, and tryptase and finely cut usingscissors, followed by reacting with each other in a shaking cultureequipment at 37 and 60 rpm for 60 minutes to separate the cells. Then,the separated cells were cultured in Dulbecco's Modified Eagles (DME)media. After culturing, the cultured cells were proliferated up topassage 3 to 4, and then the immuno-staining was performed usinganti-smooth muscle actin Ab (anti-SMC Ab) to confirm that theproliferated cells were vascular smooth muscle cells. The confirmedcells were used in the subsequent experiments. The metal plate coatedwith β-gal gene was positioned in a 12-well plate, and the vascularsmooth muscle cells were cultured thereon at 5×10⁴/well using DME mediacontaining 10% fetal bovine serum (FBS). After 7 days of culture, themetal plate in which the cells were cultured to thereby be adheredthereto was picked out and fixed, and then the staining was performed bythe same method as the X-gal staining method in Experimental Example 2.

The stained metal pieces were observed under a microscope. As a result,as shown in FIG. 12 (photograph showing that when porcine coronaryvascular smooth muscle cells were cultured on the metal pieces coatedwith the titanium dioxide/abciximab/plasmid composite layer, the geneswere transfected into the cells), it may be confirmed that the cells(arrow) adhered to the surface of the metal was observed as a bluestained cell. The result indicates that the metal/titaniumdioxide/drug/gene complex may transfect the gene in the vascular smoothmuscle cells.

The invention claimed is:
 1. A method for fabricating a gene deliverystent using titanium oxide thin film coating, the method comprising: a)a titanium oxide coating step of coating a surface of a metal stent witha titanium oxide thin film consisting of titanium oxide (TiO₂),TiO_(2-x)N_(x) wherein 0.001<x<1, or a mixture thereof; b) a surfacemodifying step of modifying the surface of the coated titanium oxidethin film to introduce a hydroxyl group; c) a drug adhering step ofadhering drugs onto the titanium oxide thin film surface-modified bybinding a functional group of the drug to the hydroxyl group of thetitanium oxide thin film to form a drug layer; and d) an oligonucleotideadhering step of adhering oligonucleotide onto the drug layer by bindingoligonucleotide to the drug to form an oligonucleotide layer wherein instep b), the surface of the titanium oxide thin film is modified for 10minutes to 2 hours by transferring water vapor, or oxygen and hydrogeninto a plasma vacuum chamber and generating non-thermal plasma, andwherein applied discharge power is in the range of 20-50 W, wherein instep b), when water vapor is used, the water vapor is transferred froman external introduction tube connected to the plasma vacuum chamberinto the plasma vacuum chamber at a reduced pressure of 1×10⁻³ to 1torr, wherein in step a) the titanium oxide thin film is coated onto thesurface of the metal stent for 1 to 6 hours by transferring a titaniumprecursor, carrier gas, and reaction gas and generating plasma in aplasma vacuum chamber, wherein the carrier as is at least one selectedfrom the group consisting of nitro en argon, and helium, wherein thereaction gas is at least one selected from the group consisting of watervapor, ozone, and oxygen, and wherein as the drug in step c), at leastone of abciximab, alpha lipoic acid, and heparin is selected to therebybe adhered.
 2. The method of claim 1, wherein the metal stent is made ofstainless steel, nitinol, tantalum, platinum, titanium, cobalt,chromium, a cobalt-chromium alloy, or a cobalt-chromium-molybdenumalloy.
 3. The method of claim 1, wherein the titanium precursor is atleast one selected from a group consisting of titanium butoxide,tetra-ethyl-methyl-amino-titanium, titanium ethoxide, titaniumisopropoxide, and tetra-methyl-hepta-diene-titanium.
 4. The method ofclaim 1, wherein the oligonucleotide in step d) is selected from a groupconsisting of gDNA, cDNA, pDNA, mRNA, tRNA, rRNA, siRNA, miRNA, andantisense-oligonucleotide.
 5. The method of claim 1, wherein in theoligonucleotide adhering step (step d), a functional group of theoligonucleotide is adhered to the functional group of the drug by atleast one bond of a hydrogen bond, a dipole-dipole bond, an induceddipole bond, and a disulfide bond (S—S bond) therebetween.